Vol 1, Issue 1, 2013

Review Article

A REVIEW ON COMMONLY USED BIOCHEMICAL TEST FOR BACTERIA

VASHIST HEMRAJ*, SHARMA DIKSHA, GUPTA AVNEET
1Department

of Pharmacy, L.R.Institute of Pharmacy, Solan (H.P), India, 173212, Email: Shimla_pharmacy@rediff.com

Received: 16 April2013 Revised and Accepted: 5 May 2013

ABSTRACT
Bacteria are meant to be omnipresent and are mostly involved in lots of human microbial infections. Such bacterial infections can be identified by
the different properties of this microorganism. Bacteria are bearing several inherent properties. by using these properties we can differentiate, can
check there presence and absence, can check their gram negative and gram positive nature and many more. The present review is therefore focused
to combine different biochemical test in one article.
Keywords: Microorganism, Bacteria, omnipresent, biochemical test.
INTRODUCTION
Different biochemicals tests are are performed to check and
differentiate the bacteria. Different biochemical test has been
mentioned below1. IMViC,
2. Sulphate reduction test.
3. Catalase production test
4. Urease test
5. Fermentation of carbohydrates
6. Starch hydrolysis test
7. Casein hydrolysis
8. Gelatin liquefaction test
9. Oxidae test
10. Microbial assay of antibiotics.
IMViCThis test is important for coliform group of bacteria. The coliform
group of bacteria includes all the aerobic and facultatively aerobic
bacteria, gram-negative nonsporulating bacilli that produce acid and
gas from the fermentation of lactose. The classical species of this
group are Escherichia coli and Enterobacter aerogene. The
relationship of these organisms to others of the enteric group is with
salmonella, shigella, Klebsiella, proteus, Serratia and other genera
which are Gram negativep. The IMViC name stands for the first letter
of each test in the series with the lower i for case for pronunciation
the IMViC test is designated to differentiate E.coli from Enterobacter
aerogenes1.

As far back as 1889, the indole test was used as a means to

distinguish between Escherichia coliand Enterobacter aerogenes 2.
The numerous variations of the indole test alone and in combination
with other biochemical tests attest to the central role this test has
played in the characterization of coliforms (gram-negative
nonsporulating bacilli that ferment lactose, producing acid and gas)3.
The indole test is still used as a classic test to distinguish . Indole
positive E coli/from enterobactor and klebsiella 4. The methyl red
test (MR) was developed by Clark and Lubes in 1915 who rather
optimistically laid that simple condition have been found under
which metabolism can be so controlled that the hydrogen ion
concentration of culture of one group can be made to diverge widely
from those of the other group.
INDOL TEST
Purpose
The indole test screens for the ability of an organism to degrade the
amino acid tryptophan and produce indole. It is used as part of the
IMViC procedures, a battery of tests designed to distinguish among
members of the family Enterobacteriaceae.
Principle
Tryptophan is an essential amino acid, which is oxidized by some
bacteria resulting in the formation of indole, pynivic acid and
ammonia. The indole test is done by inoculating the test organism
into tryptophan broth, which contain tryptophan. The indole which
is produced is detected by adding KOVACs reagent which produced
cherry red colored ring.

Vashist hemraj et al.

Innovare Journal of Life Science, Vol 1, Issue 1, 2013, 1-7

Figure.1: Shows Methyl red test

Dissolve the ingredients in 1 liter of sterile water. Dispense 4 ml per
tube. Cap tube and autoclave at 121oC under 15 psi pressure for 15
minutes. Store the tubes in the refrigerator at 4 to 10C.

SIM medium
Ingredients

Amount

Ingredients of KOVACS reagent5-6.

Peptone
Beef extract

30.0 g
3.0 g
0.2 g

Ingredient

Amount

Amyl or isoamyl,reagent grade

(Butyl alcohol may be substituted.
P-dimethylaminobenzaldehyde (DMAB)
HCL (concentrated)

150.0ml
10.0ml
50.0ml

Dissolve DMAB in the alcohol. Gentle heating might be required to

get the aldehyde into solution. Slowly add the acid to the aldehydealcohol mixture. The solution should be a pale yellow color and is
only stable for a short time. Store the mixture in a brown glass
bottle in the refrigerator.
Procedure to perform the testTrypton broth was prepared according to the given composition.
The broth was dispensed into the tubes and sterilized. The test
organisms were inoculated into the tubes and one was left
uninoculated as control. The tubes were inoculated at 37oc for 48h
After incubation 1ml of KOVACs reagent was added to all the tubes
including control. The tubes were shaken gently and allowed to
stand for 1-2 min. The tubes were observed for formation of cherry
red ring.
Alternate Methods of Detecting Indole Production Tryptophan
peptone broth
Tryptophan peptone broth 7- 8
Ingredients
Casein peptone
Sodium chloride
Tryptophan

Amount
10.0 g
5.0 g
100 g

The tryptophan concentration can be varied; the amount used here

will result in a 1% final concentration. Dissolve ingredients in 1 liter
of distilled water. Dispense 4 ml per tube. Cap tube and autoclave at
121C under 15 psi pressure for 15 minutes.Store tubes in the
refrigerator at 4 to 10C. Inoculate, incubate, and perform test as
described above for tryptone broth.
Sulfide-indole-motility (SIM) medium7-9 The SIM medium is a
multitest agar used to test for indole production while
simultaneously determining other characteristics of the Bacterium .

Ferrous ammonium sulfate

Sodium thiosulfate
Agar

0.025 g
3g

MIO medium
Ingredients
Yeast extract
Peptone
Tryptone
L-ornithine HCl
Dextrose
Agar
Bromcresol purple

Amount
3.0 g
10.0 g
10.0 g
5.0 g
1.0 g
2.0 g
0.02 g

Dissolve ingredients, except agar, in 1 liter of distilled water. Adjust

pH to 7.3. Add agar and heat mixture to boiling to dissolve agar.
Cool to 50C. Dispense in 4.0 to 5.0 ml aliquots in 16-mm test tubes.
Cap tubes and autoclave at 121C under 15 psi pressure for 15
minutes. After autoclaving, allow tubes to cool in an upright position
to form the agar deep. Tubes can be stored at 4 to 8C for several
months. SIM medium is commercially available both as a premixed
powder and as premade deep tubes. To inoculate SIM medium, pick
an isolated colony with a needle. Stab the needle approximately twothirds of the way into the deep and then remove it following the
same path as the entry. Incubate at 35C (+/-2C) for 24 to 48 hours
or until growth is evident. To test for the presence of indole, a byproduct of tryptophan metabolism, 5 drops of Kovcs reagent should
be
added
to
the
top
of
the
deep.
A positive indole test is indicated by the formation of a red color in
the reagent layer on top of the agar deep within seconds of adding
the reagent. If a culture is indole negative, the reagent layer will
remain yellow or be slightly cloudy.See Indole Atlas images
Alternative Methods for Determining Indole Production for SIM
result images.
Motility-indole-ornithine (MIO) medium5, 7 The MIO medium is a
multitest agar used to test for indole production while
simultaneously determining other characteristics of the bacterium
(see Comments and Tips section).
Bring to 1 liter with distilled water. Heat mixture to boiling to
dissolve agar. Cool to 50C. Dispense in 4.0 to 5.0 ml aliquots in 16mm test tubes. Cap tubes and autoclave at 121C under 15 psi
pressure for 15 minutes. After autoclaving, allow tubes to cool in an
upright position to form the agar deep. Tubes can be stored at 4 to
8C for several months. MIO medium is commercially available both

Vashist hemraj et al.

Innovare Journal of Life Science, Vol 1, Issue 1, 2013, 1-7

as a premixed powder and as premade deep tubes.To inoculate MIO

medium, pick an isolated colony with a needle. Stab the needle
approximately two-thirds of the way into the deep and then remove
it following the same path as the entry. Incubate at 35C (+/-2C) for
24 to 48 hours or until growth is evident.To test for the presence of
indole, a by-product of tryptophan metabolism, 5 drops of Kovcs
reagent should be added to the top of the deep.
A positive indole test is indicated by the formation of a red color in
the reagent layer on top of the agar deep within seconds of adding
the reagent. If a culture is indole negative, the reagent layer will
remain yellow or be slightly cloudy.See Indole Atlas images
Alternative Methods for Determining Indole Production for MIO
result images.
Ehrlich's reagent9
Ehrlich's reagent, an alternative to Kovcs reagent, also contains
DMAB, which reacts with indole to produce a red product. The
Ehrlich formulation is more sensitive but contains additional toxic or
flammable solvents; it is recommended when testing bacterial
groups that produce little indole such as nonfermentative bacilli or
anaerobes. Kovcs reagent is apparently more stable and the
absence of the additional organic extraction (required with
Ehrlich's) makes Kovcs formulation more suitable for
undergraduate laboratories.

Figure.2: Shows methyl red test

Results

Ehrlichs reagent
Ingredients

Amount

Ethyl alcohol (absolute)

95.0 ml

p-dimethylaminobenzaldehyde (DMAB)
HCl (concentrated)

1.0 g
20.0 ml

METHYL RED TEST [10-12]

Theory-The test measures the final acidity of a culture in buffered
medium containing glucose and peptose, but the pH value recorded
at the time of the test is not necessarily the lowest value attained
during growth. In the early stage of glucose fermentation sufficient
acid is produced by both E.coli and Klebsiella aurogens to turn
methyl red indicator orange and red (+ve) but on further incubation
Klebsiella aurogens breaks down the Pyruvic acid and other acids
and produces a reversion in the reaction of the culture towards
neutrality; methyl red turns yellow (-ve).Timing of the sequence
Starting pH (c.7)

Low pH (c.5)

higher pH (6 or more)

Will be affected by the medium, particularly the buffering power of

the peptone and the phosphate by the temperature and duration of
incubation, and by the strain. It is surprising therefore, that a greater
attempt has not been made to standardize the test.

VOGES-PROSKAUER TEST13-15
Purpose
VogesProskauer or VP is a test used to detect acetoin in
a bacterial broth culture. The test is performed by adding alphanaphthol and potassium hydroxide to the Voges-Proskauer broth
which has been inoculated with bacteria. A cherry red color
indicates a positive result, while a yellow-brown color indicates a
negative result.
The test depends on the digestion of glucose to
acetylmethylcarbinol. If glucose is being broken down, it will react
with alpha-naphthol (VP reagent #1) and potassium hydroxide (VP
reagent #2) to form a red color. Alpha-naphthol and potassium
hydroxide are chemicals that detect acetoin.
Procedure: First, add the alpha-naphthol; then, add the potassium
hydroxide. A reversal in the order of the reagents being added may
result in a weak-positive or false-negative reaction.
VP positive organisms include Enterobacter, Klebsiella, Serratia
marcescens, Hafnia alvei, Vibrio damsela, and Vibrio alginolyticus.
History- The reaction was developed by Daniel Wilhelm Otto Voges
and Bernhard Proskauer German bacteriologist in 1898 at the
Institute of infectious Disease.

Some bacteria perform mixed-acid fermentation. The by-products

are mixtures of large amounts of stable acids. Other fermentative
organisms produce smaller amounts of less stable acids.
The Methyl-Red test tests for the ability to perform mixed-acid
fermentation. MR-VP broth contains glucose, peptone, and a
phosphate buffer. Organisms that perform mixed-acid fermentation
produce enough acid to overcome the buffering capacity of the
broth, so a decrease in pH results. Organisms that perform other
kinds of fermentation cannot overcome the buffering capacity of the
broth.
After incubation, the pH indicator Methyl Red is added to the broth.
Methyl Red is red at pH below 4.4 (this would be a positive result)
and yellow at pH above 6.0. An orange color indicates an
intermediate pH and would be considered a negative result.
This test is among a suite of tests (Indole, Methyl-Red, VoguesProskauer, and Citrate) that are used to differentiate among the
Gram-Negative bacilli in the family Enterobacteriaceae.

Figure.3: Shows Voges Proskauer test

Vashist hemraj et al.

CITRATE UTILIZATION TEST [17-18]

Purpose-The test is used to check the ability of microorganisms for
utilization of citrate.
Principle-Citrate test is used to differentiate among enteric bacteria
on the basis of their ability to utilize as their soul carbon source. The
utilization of citrate depends upon the presence of enzyme Citrate
Permease produced by organism that helps its transports into the
cell.
The citrate test is performed by inoculating microorganism into in
an organic synthetic media, Simons Citrate broth when sodium
citrate is only source of Carbon and energy.Bromothymol blue is
used as an indicator when the citric acid is metabolized
,carbondioxide is generated which combines withsodium and water
to form sodium carbonate which is an alkaline product which is
responsible for change in colour from green to blue and this
constitute positive test.

Innovare Journal of Life Science, Vol 1, Issue 1, 2013, 1-7

Procedure- Prepare the sulphate agar according to given

composition . sterilize the Sulphate agar by autoclaving at 121oc at
15 lbs pressure for 20 minutes. Dispense the sterilized agar into
sterilized test tubes and labeled with respective organism. Stab
inocculate the test organism after solidification of the medium and
incubate the tubes at 37oc for 24 hrs and observed for black
coloration at the point of stab.
CATALASE PRODUCTION TEST20
Purpose- The test is done to check weather the test organism
produce Catalase or not.
Principle-The oxidation of flavoproteins invariably result in the
formation of Hydrogen peroxide as one major product. In addition
this oxidation (and other oxygenation) produce small quantities of
and even more toxic radical-The superoxide
In aerobes and aerotolerant aerobes the potentially leathal
accumulation of oxygen is prevented by the enzyme superoxide
dismutase which catalysis it to hydrogen peroxide and oxygen.
2e-

2H+

2O2

2O

O 2+H2O2
Superoxide mutase

H2O2 is produced ois lethal to the cells and Catalase breaks it to

water and oxygen .
2H2O2

O2+ 2H2O

Catalase

Figure 4: Shows Citrate utilization test.

In this test the organism is subjected to 3% H2O2 solution and

Catalase enzyme acts on it just as it could H2O2 is produced inside
the cell. Catalase lies close to the cell membrane.A positive result is
detected by the formation of air bubbles.

Procedure-Prepare the citrate slant according to the given

composition and dispensed into the the tes tube and autoclaved at
121oc for 15lbs.inocciulate the tubes with the given culture and then
inoculate at 37oc for 48 hours. Observe the tubes for colour change
from green to blue.
SULPHATE REDUCTIOIN TEST19Purpose- The test is done to show the presence of hydrogen
sulphide by sulphide reducing bacteria.
Principle- H2S is formed by some bacteria by reduction of sulphure
containing amino acids (Cystein), Cysteine and metheonine or
through reduction of inorganic sulphure compounds like
thiosulphates (S2O3-)or sulphates (SO4 --) or sulphite (SO3-- ).The H2S
production can be detected by incorporating a heavy metal salt
containing ion (Fe++) or lead (Pb++) ion as a H2S indicator to
neutrient
culture
medium
containing
cystein
and
sodiumthiosulphate as the sulphure substance.H2S a colourless gas
when produced react with metal salts (FeSO4) forming visible
insoluble black Ferrous Sulphide precipitates. Production of H 2S
from systein and Na2S2O3.

Figure.6: Shows Catalase production test

Procedure- Take a drop of reagent on a clean slide and transfer
bacterial colonies into it. Observe the effervescence formation.
UREASE TEST21-22Purpose-the test is done if the given organism produces enzyme
Urease or not.
Pinciple-Urea is a major organic waste production of protein
digestion by most vertebrates and is excreted in urine.Some
microorganisms have the ability to produce the enzyme urease.
Urease is a hydrolytic enzyme, which attack the amide linkage
liberating Ammonia.
2HN

C = O + 2H2O

Figure.5: Shows Sulphate reduction test

2HN

2NH3 + H2O
Amonia

Vashist hemraj et al.

Innovare Journal of Life Science, Vol 1, Issue 1, 2013, 1-7

This test distinguishes members of genus Proteus from other lactose

non fermenting enteric microbes. Urease test is performed by
growing test organisms on urea broth or agar medium containing P H
6.8 indicator phenol red. During incubation microorganism
processing urease will produce ammonia that raises the P H of the
medium (PH 8.1 or >)

Figure.8: Shows fermentation of carbohydrates

Procedure
Figure.7: Showing Urease test
Procedure- Prepare and dispense Urea agar ( basal medium) into
tubes and sterilized.Glucose and phenol red is added to the basal
medium and steamed for 1 Hr.Add filtered sterilized urea solution
and mix all contents well and dispense into sterile test tubes.The test
organisms are inoculated and incubated at 37oc for 24-48 Hrs.
Colour change is observed.
FERMENTATION OF

CARBOHYDRATE23:

Purpose- This test I performed to check the ability of bacteria to

check its ability to ferment sugar.
Introduction-: Morphological and cultural characteristics are
essential in identifying an organism in the case of bacteria.
Physiological information (through physiological reactions) in an
organism contribute informations for specific identification as all the
biochemical reactions are catalysed by enzyme and enzyme specific
to species the bio-oxidation reaction could be oxidative or
fermentative oxidizing organic substance s for energy result in the
production of CO2 and water it is oxidative reaction.Aldehydes or
alcohols produced with various type of gases such as CO 2,H2,CH4s
called fermentive reactions.Fermentation of sugars can be easily
demonstrated and the end products of particular fermentation
depends on the nature of the organism characteristics of substrate
and environmental conditions such as temperature and PH.
Principle- Fermentation can be defined as an energy yielding
process that does not involve an electron transport chain and
exogenous terminal electrons acceptors instead it relies on substrate
level. Phasphorylation and an endogenously generated electron
acceptor.

The fermentation medium is prepared and sterilized with

the indicator and Durhams tube has no air bubbles in
them.
The sugar solution is autoclaved at 10 lbs/Sq inch
pressure for 10 minutes and 0.5 ml of the sugar is added
to sterile peptone water.
The fermentation tubes are inoculated with the test
organism.
Negative control is maintained for all the sugars.
The tubes are inoculated at 37oc for 24 hrs.

STARCH HYDROLYSIS 24Purpose-This test is performed to test the utilization of starch by

Bacteria by producing the enzyme Amylase.
Principle: Amylase is an exoenzyme that hydrolysis starch, a
polysaccharides into monosaccharide and disaccharides such as
glucose.these mono and disaccharide enters into cell cytoplasm of
bacteria through the semi-permeable membrane and their by
attacked by endoenzymes.Starch is a complex carbohydrate
composed of glucose molecules linked together by -1,4 and -1,6
glycosidic bonds.
Amylase production is known in some bacteria while well known
known in some fungi.
The ability to degrade starch is used as the criteria for determination
of amylase production by a microbe. In laboratory it is tested by
performing the agar test to determine the absence and presence of
iodine produces a dark blue coloration and a yellow zone around the
colony in an otherwise blue media indicates amylolytic activity.

Fermentation of sugar is carried out anaerobically with the indicator

production incompletely oxidized acidic or alkaline and products
with gas production, to detect fermentation in labs. A fermentation
tube is used with a Durhams tube for the detection of gas as the end
product of carbohydrate metabolism.
Fermentation broth contains a nutrient broth with specific
carbohydrates may be altered during autoclaving by heat in the
pressure of other ingredients so they are sterilized separately.
Peptone water base is also autoclaved separately at 121 oc. for 15
minutes.

Figure.9: Shows starch hydrolysis

Vashist hemraj et al.

Procedure

Innovare Journal of Life Science, Vol 1, Issue 1, 2013, 1-7

Procedure

Sterile starch agar medium is poured on to the sterile

petriplates and allowed to solidify.
The test organism is streaked on the plate and incubated for
48 hrs. at 37oc.
The plates are flooded with grams iodine and excess iodine
is drained off.
Plate are examined for the zone of clearance around the
growth for each organism.

Prepare the media according to the given composition and

sterilize after pouring into the test tubes.
Inoculate the test organism into the sterilized test tubes
and left one uninocculated as control .
Incubate the tube at 37oc for 24-48 hrs.
After incubation kept the tubes in an ice bath for 30 min.
note down the results depending on the condition
wheather gelatin is in the liquid state or in the solid form

CASEIN HYDROLYSIS [25-26]

Purpose- This test is done to check the ability of microorganism to
hydrolyse casein .

Figure.11: Showing Gelatin liquification test

OXIDASE TESTFigure 10. Shows casein hydrolysis
Principle-Casein is a major protein found in milk. It is a
macromolecule composed of amino acid linked together by peptide
bond. Some microorganism have the ability to decrease the protein
casein by producing proteolytic exo-enzymes called protease. The
process breake down the peptide bond by introducing water into the
molecule liberating the soluble amino acid pool for use in the
synthesis of structural and functional cellular protein.
Casein hydrolysis can be demonstrated by supplementing nutrient
agar media with milk .The medium is opaque due to the casein is
colloidal suspension formation of a clear zone surrounded the
bacterial growth. after inoculation and incubation of agar plate
culture is due to hydrolysis of casein by protease activity. The
medium surrounding the growth of the organism remains opaque
since it defects light rays rather than transmitting which indicates a
negative reaction.
Procedure
1. Prepared the Casein media.
2. Sterilize the media by autoclaving.
3. Then dispense the media in sterilized petriplates and after
solidification streake the test organism at the centre of the plate.
4. incubate the plate at 37oc for 24 hrs.
5. After incubation the plates are observed for hollow and clear zone
around the colonies.

Purpose-The test is done to detect the presence of cytochrome C

and hence the production of oxidase enzyme by given test organism.
Principle-Cytochrome are heam containing catalytic enzyme which
are tightly bound to (prokaryotic cells) cells of plasma
membrane.they are concerned with later stages of biochemical
oxidation.They act as electron or H2 carries of biological oxidation by
virtue of their availability valence by heam ions i.e., they can
undergo reduction and oxidation . During period of great activity
they are reduced. Cytochrome C is more abundant and freely soluble
in water. Cytochrome C does not react directly with O2 but reduced
form will be oxidized by cytooxidase with which it is closely
associated. Cyto a,a3 is called cytooxidase. or indophenolase or
endophenol oxidase or ferro cytochrome C or oxygen oxido
reductase.
Cyt. a.a3 is the terminal codon in electron transport chain hence
called Cytooxidase.This test tests for lytic and not cytooxidase.
In this test an oxidisable amine namely tetramethyl-p-phenylene
diamine dihydrochloride is used to detect cytochrome C as wel as
oxidase enzyme on flooding of bacterial cell with reagent, a purple
colour is formed due to oxidation of reagent. Eletrones are
transferred to cytochrome C and from cytochrome c to molecular
oxygen via cytochrome a,a3 .The enzyme catalyses the transfer of
electrons from reagent to molecular oxygen.

GELATIN LIQUIFICATION TEST [27-33]

Purpose-The test is performed to check the ability of the
microorganism to produce the enzyme gelatinase.
Principle-Gelatin is a protein derived form collagen,which is
insoluble in cold water but insoluble in cold water but soluble in hot
water and form gel on cooling it. gelatin is liquid at room
temperature that solidify on cooling up to the temperature of -4oc for
bacteriological use and edible grade of gelatin is used since it is free
of preservatives and inhibitory amount of heavy metals.
The proteolytic organisms digest protein and may liquefy gelatin. the
liquification of gelatin is index of proteolytic activity of the organism
produce the enzyme gelatinase.
Figure. 12 : Showing Oxidase test

Vashist hemraj et al.

PROCEDURE

Place 1-2 drops of 1 % oxidase reagent on 6 cm square

piece of whatman filter paper.
Transferr a small colony of test organism using a loop onto
soaked filter paper and observe for purple colour
development.
A positive test is indicated by development of purple colour
in 5-10 seconds.

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